1) Field of the Invention
Embodiments of the invention are most generally related to the field of multiphoton fluorescence and/or non-linear harmonic emission endoscopy apparatus and methods. More particularly, embodiments of the invention are directed to multi-magnification, non-confocal, multi-path optical systems and optical system modules for use with fluorescence emission endoscopy systems, and associated methods.
2) Description of the Related Art
The multiphoton microscope was co-invented almost two decades ago by Dr. Watt Webb, a co-inventor of the present invention. Multiphoton microscopy (MPM), as is now well known, is a special kind of laser scanning microscopy that provided significant advantages over standard confocal microscopy. In confocal microscopy, one photon of high energy light (at, e.g., 488 nm) is used to excite a molecule to produce one photon of fluorescence. The light excites molecules in a relatively large region around the focal point. The use of high energy light could easily damage living tissue in the entire region of exposure. Furthermore, imaging depth was limited to about 50 microns (μ) (about five cell layers).
In MPM, multiple low energy photons (at, e.g., 960 nm) impinge on a fluorescent molecule simultaneously, producing one photon of fluorescence from the focal volume of the excitation field. Advantageously, MPM is safer and more efficacious than confocal microscopy for human use because of, e.g., limited site photo-toxicity and photo-damage to living tissue, imaging depths up to 500 to 1000 μ, and lower out-of-focus fluorescence background. Intrinsic fluorescence of certain tissue structures generated by the excitation field reduces or eliminates the need for dye (fluorophore) injection. There are other reasons known in the art. As a result, MPM provides the capability to acquire high contrast, high resolution images, without the need to use pinhole apertures or other spatial filtering elements, with reduced tissue photo-bleaching and photo-destruction that occur from repeated excitation.
The laser light used to generate multiphoton excitation also supports the non-linear optical phenomenon known as harmonic generation. Second harmonic generation (SHG) (and higher-order harmonic generation) under multiphoton excitation can cause collagen and certain tissue structures such as microtubule bundles, nerves and cartilage, for example, to emit intrinsic SHG radiation.
The present inventors, and others, have recognized that various advantages and benefits could be realized by incorporating the principles of MPM into an endoscope. For example, disease diagnosis has for a long time been, and continues to be, carried out by various biopsy procedures. A biopsy requires the physical removal of a (deep) tissue sample from a patient, sample analysis by a pathologist, and reporting, which may take from a few hours to several days or more. The ability to perform real time, in-situ endoscopy in combination with the diagnostic capabilities of multiphoton (and/or harmonic generated) fluorescence imaging could significantly reduce the pain, time, and cost associated with conventional biopsy procedures and assist in disease diagnosis and the extent of tissue damage due to disease states. High resolution MPM endoscopy for sub-tissue, nerve, and cartilage examination offers advantages over the capabilities of current surgical endoscopes. The ability to see nerves and collagen clearly would be especially valuable, for example, in nerve-sparring prostate surgery, bladder cancer treatment, and in maxillofacial and oral surgery.
The simultaneous demands of low magnification, large field of view imaging and high magnification, high-resolution multiphoton imaging necessitate two effective optical imaging systems (e.g., two objective lenses). Although multi-optical systems are routinely provided in microscopy apparatus, separate, switchable optical systems do not provide a suitable architecture for a compact endoscope.
Efforts to date to improve endoscopic imaging procedures and apparatus have focused on the multiphoton fluorescence excitation processes with little attention directed to improved systems and methods for acquiring, identifying, and analyzing the fluorescence, or to improved systems and methods to reduce the severity and invasiveness of existing procedures.
In view of the aforementioned challenges and shortcomings associated with fluorescence emission endoscopy imaging apparatus and methods, the inventors have recognized the unfilled need for apparatus and methods that can address these challenges and shortcomings, and others, in a practical, cost effective, and efficacious manner.
The inventors have also recognized that conventional confocal-based endoscopy imaging apparatus and methods can be disadvantageous due, for example, to image obstruction from scanner apparatus and inefficient signal collection and/or transmission via a concentric excitation fiber waveguide.
Embodiments of the invention are directed to apparatus and methods that address the foregoing mentioned shortcomings and disadvantages associated with current technology in this field.